1 Instructor names and contact information
- Benjamin P. Berman (Benjamin.Berman at cshs.org)
- Tiago C. Silva (tiagochst at gmail.com)
2 Workshop description
2.1 Aims
This workshop will:
- Introduce you to the TCGA (The Cancer Genome Atlas) data available at the NCI’s Genomic Data Commons (GDC).
- Demonstrate how to access and import TCGA data using the R/Bioconductor package TCGAbiolinks (Colaprico et al. 2015)
- Provide intruction to visualize copy number alteration and mutation data using the R/Bioconductor package maftools
2.2 Useful links
- the NCI’s Genomic Data Commons (GDC): https://gdc.cancer.gov/.
- TCGAbiolinks package: http://bioconductor.org/packages/TCGAbiolinks/
- maftools package: http://bioconductor.org/packages/maftools/
2.3 Reference articles
- TCGAbiolinks:
- Colaprico, Antonio, et al. “TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data.” Nucleic acids research 44.8 (2015): e71-e71. Silva, Tiago C., et al. “TCGA Workflow: Analyze cancer genomics and epigenomics data using Bioconductor packages.” F1000Research 5 (2016). (https://f1000research.com/articles/5-1542/v2)
- Mounir, Mohamed, et al. “New functionalities in the TCGAbiolinks package for the study and integration of cancer data from GDC and GTEx.” PLoS computational biology 15.3 (2019): e1006701. (https://doi.org/10.1371/journal.pcbi.1006701)
- Silva TC, Colaprico A, Olsen C et al.TCGA Workflow: Analyze cancer genomics and epigenomics data using Bioconductor packages [version 2; peer review: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:1542 (https://doi.org/10.12688/f1000research.8923.2).
- The NCI’s Genomic Data Commons (GDC):
- Gao, Galen F., et al. “Before and After: Comparison of Legacy and Harmonized TCGA Genomic Data Commons’ Data.” Cell systems 9.1 (2019): 24-34. (https://doi.org/10.1016/j.cels.2019.06.006)
2.4 R/Bioconductor packages used
3 Introduction
3.1 Understanding GDC (genomics data common portal)
In this workshop we will access (The Cancer Genome Atlas) data available at the NCI’s Genomic Data Commons (GDC). Before we start, it is important to know that the data is deposit in two different databases: - The legacy archive (https://portal.gdc.cancer.gov/legacy-archive/search/f) which contains unharmonized legacy data from repositories that predate the GDC (e.g. CGHub). Also, legacy data is not actively maintained, processed, or harmonized by the GDC (Source: https://docs.gdc.cancer.gov/Data_Portal/Users_Guide/Legacy_Archive/)
- Harmonized database (https://portal.gdc.cancer.gov/) which contains data from many cancer projects processed using standardized pipelines and the reference genome GRCh38 (hg38). This gives the advantage of analyzing multiple cancer types or the same cancer type across multiple projects. You can find more information about the pipelines supporting data harmonization at https://gdc.cancer.gov/about-data/gdc-data-harmonization and at https://docs.gdc.cancer.gov/Encyclopedia/pages/Harmonized_Data/.
An overview of the web portal is available at https://docs.gdc.cancer.gov/Data_Portal/Users_Guide/Getting_Started/.
Figure legend: TCGA databases (blue: legacy archive. red: harmnized database) Source: https://portal.gdc.cancer.gov/
3.2 Understanding TCGA data
3.2.1 Data access
GDC provides the data with two access levels:
- Open: includes high level genomic data that is not individually identifiable, as well as most clinical and all biospecimen data elements.
- Controlled: includes individually identifiable data such as low level genomic sequencing data, germline variants, SNP6 genotype data, and certain clinical data elements
You can find more information about those two levels and how to get access to controlled data at: https://gdc.cancer.gov/access-data/data-access-processes-and-tools.
3.2.2 TCGA barcode description
Each TCGA sample has a unique identifier called TCGA barcode, which contains important information about each sample. A drescription of the barcode is shown below (Source: https://docs.gdc.cancer.gov/Encyclopedia/pages/TCGA_Barcode/).
Figure legend: TCGA barcode Source: https://docs.gdc.cancer.gov/Encyclopedia/pages/TCGA_Barcode/
You can find a table with all the code and meanings at https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables. The Sample Type Codes is shown below:
Figure legend: TCGA samples type. Source: https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/sample-type-codes
3.2.3 Data structure
In order to filter the data available in GDC some fields are available such as project (TCGA, TARGET, etc), data category (Transcriptome Profiling, DNA methylation,Clinical, etc), data type (Gene Expression Quantification, Isoform Expression Quantification, Methylation Beta Value, etc), experimental strategy (miRNA-Seq, RNA-Seq, etc,), Workflow Type, platfom, access type and others.
Figure legend: Data category filter
Figure legend: Data type filter
In terms of data granularity, a project has data on several categories, each category contains several data types that might have been produced with different workflows,experimental strategy and platforms. In that way, if you select data type “Gene Expression Quantification” the data category will be Transcriptome Profiling.
Figure legend: Filtering by Gene Expression Quantification
You can find the entry possibilities for each filter at the repository page of the database at https://portal.gdc.cancer.gov/repository.
4 Working with TCGA data
During this workshop we will be using open access data from the harmonized GDC database (hg38 version), which can be accessed at https://portal.gdc.cancer.gov/.
Through the next sections we will:
- Access TCGA clinical data
- Search, download and make an R object from TCGA data
- RNA-seq data
- DNA methylation
- Download samples metadata
- Download and visualize mutations
- Download and visualize copy number alterations
4.3 RNA-Seq data
The RNA-Seq pipeline produces raw counts, FPKM and FPKM-UQ quantifications and is described at https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/.
Figure legend: gene expression pipeline. Souce: https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/
Figure legend: mRNA files. Souce: https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/
The following options are used to search mRNA results using TCGAbiolinks:
- data.category: “Transcriptome Profiling”
- data.type: “Gene Expression Quantification”
- workflow.type: “HTSeq - Counts”, “HTSeq - FPKM”, “HTSeq - FPKM-UQ”
Here is the example to download the raw counts, which can be used with (http://bioconductor.org/packages/DESeq2/) for differential expression analysis.
query.exp.hg38 <- GDCquery(project = "TCGA-GBM",
data.category = "Transcriptome Profiling",
data.type = "Gene Expression Quantification",
workflow.type = "HTSeq - Counts",
barcode = c("TCGA-14-0736-02A-01R-2005-01", "TCGA-06-0211-02A-02R-2005-01"))
GDCdownload(query.exp.hg38)
raw.counts <- GDCprepare(query = query.exp.hg38, summarizedExperiment = FALSE)## # A tibble: 6 x 3
## X1 `TCGA-14-0736-02A-01R-2005-… `TCGA-06-0211-02A-02R-2005…
## <chr> <dbl> <dbl>
## 1 ENSG00000000003… 4282 3853
## 2 ENSG00000000005… 34 8
## 3 ENSG00000000419… 899 1785
## 4 ENSG00000000457… 293 551
## 5 ENSG00000000460… 143 378
## 6 ENSG00000000938… 286 683
Here is the example to download the FPKM-UQ counts.
query.exp.hg38 <- GDCquery(project = "TCGA-GBM",
data.category = "Transcriptome Profiling",
data.type = "Gene Expression Quantification",
workflow.type = "HTSeq - FPKM-UQ",
barcode = c("TCGA-14-0736-02A-01R-2005-01", "TCGA-06-0211-02A-02R-2005-01"))
GDCdownload(query.exp.hg38)
fpkm.uq.counts <- GDCprepare(query = query.exp.hg38, summarizedExperiment = FALSE)## # A tibble: 6 x 3
## X1 `TCGA-14-0736-02A-01R-2005-… `TCGA-06-0211-02A-02R-2005…
## <chr> <dbl> <dbl>
## 1 ENSG00000242268… 1533. 3518.
## 2 ENSG00000270112… 4253. 4978.
## 3 ENSG00000167578… 261187. 135247.
## 4 ENSG00000273842… 0 0
## 5 ENSG00000078237… 224251. 180270.
## 6 ENSG00000146083… 117081. 197064.
# miRNA
library(TCGAbiolinks)
query.mirna <- GDCquery(project = "TARGET-AML",
experimental.strategy = "miRNA-Seq",
data.category = "Transcriptome Profiling",
barcode = c("TARGET-20-PATDNN","TARGET-20-PAPUNR"),
data.type = "miRNA Expression Quantification")
GDCdownload(query.mirna)
mirna <- GDCprepare(query = query.mirna,
save = TRUE,
save.filename = "mirna.rda")
query.isoform <- GDCquery(project = "TARGET-AML",
experimental.strategy = "miRNA-Seq",
data.category = "Transcriptome Profiling",
barcode = c("TARGET-20-PATDNN","TARGET-20-PAPUNR"),
data.type = "Isoform Expression Quantification")
GDCdownload(query.isoform)
isoform <- GDCprepare(query = query.isoform,
save = TRUE,
save.filename = "mirna-isoform.rda")
DNA methylation: aligned against hg38
#--------------------------------------
# DNA methylation data
#--------------------------------------
# DNA methylation aligned to hg38
query_met.hg38 <- GDCquery(project= "TCGA-LGG",
data.category = "DNA Methylation",
platform = "Illumina Human Methylation 450",
barcode = c("TCGA-HT-8111-01A-11D-2399-05","TCGA-HT-A5R5-01A-11D-A28N-05"))
GDCdownload(query_met.hg38)
data.hg38 <- GDCprepare(query_met.hg38)4.4 Mutation
TCGAbiolinks has provided a few functions to download mutation data from GDC. There are two options to download the data:
- Use
GDCquery_Mafwhich will download MAF aligned against hg38.
This example will download MAF (mutation annotation files) for variant calling pipeline muse. Pipelines options are: muse, varscan2, somaticsniper, mutect. For more information please access GDC docs.
##
## Attaching package: 'dplyr'
## The following object is masked from 'package:matrixStats':
##
## count
## The following object is masked from 'package:Biobase':
##
## combine
## The following objects are masked from 'package:GenomicRanges':
##
## intersect, setdiff, union
## The following object is masked from 'package:GenomeInfoDb':
##
## intersect
## The following objects are masked from 'package:IRanges':
##
## collapse, desc, intersect, setdiff, slice, union
## The following objects are masked from 'package:S4Vectors':
##
## first, intersect, rename, setdiff, setequal, union
## The following objects are masked from 'package:BiocGenerics':
##
## combine, intersect, setdiff, union
## The following objects are masked from 'package:stats':
##
## filter, lag
## The following objects are masked from 'package:base':
##
## intersect, setdiff, setequal, union
## ============================================================================
## For more information about MAF data please read the following GDC manual and web pages:
## GDC manual: https://gdc-docs.nci.nih.gov/Data/PDF/Data_UG.pdf
## https://gdc-docs.nci.nih.gov/Data/Bioinformatics_Pipelines/DNA_Seq_Variant_Calling_Pipeline/
## https://gdc.cancer.gov/about-gdc/variant-calling-gdc
## ============================================================================
## --------------------------------------
## o GDCquery: Searching in GDC database
## --------------------------------------
## Genome of reference: hg38
## --------------------------------------------
## oo Accessing GDC. This might take a while...
## --------------------------------------------
## ooo Project: TCGA-COAD
## --------------------
## oo Filtering results
## --------------------
## ooo By access
## ooo By data.type
## ooo By workflow.type
## ----------------
## oo Checking data
## ----------------
## ooo Check if there are duplicated cases
## ooo Check if there results for the query
## -------------------
## o Preparing output
## -------------------
## Downloading data for project TCGA-COAD
## Of the 1 files for download 1 already exist.
## All samples have been already downloaded
##
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## -Validating
## -Silent variants: 77997
## -Summarizing
## --Mutiple centers found
## BCM;WUGSC;WUGSC;BCM;BCM;BI--Possible FLAGS among top ten genes:
## TTN
## MUC16
## SYNE1
## OBSCN
## -Processing clinical data
## --Missing clinical data
## -Finished in 11.5s elapsed (12.0s cpu)
datatable(getSampleSummary(maf),
filter = 'top',
options = list(scrollX = TRUE, keys = TRUE, pageLength = 5),
rownames = FALSE)4.5 Copy number alteration data
The Copy Number Variation Analysis Pipeline is described at https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/CNV_Pipeline/
Numeric focal-level Copy Number Variation (CNV) values were generated with “Masked Copy Number Segment” files from tumor aliquots using GISTIC2 on a project level. Only protein-coding genes were kept, and their numeric CNV values were further thresholded by a noise cutoff of 0.3:
- Genes with focal CNV values smaller than -0.3 are categorized as a “loss” (-1)
- Genes with focal CNV values larger than 0.3 are categorized as a “gain” (+1)
- Genes with focal CNV values between and including -0.3 and 0.3 are categorized as “neutral” (0).
You can access “Gene Level Copy Number Scores” from GISTIC with the code below:
query <- GDCquery(project = "TCGA-GBM",
data.category = "Copy Number Variation",
data.type = "Gene Level Copy Number Scores",
access = "open")
GDCdownload(query)
scores <- GDCprepare(query)## # A tibble: 5 x 5
## `Gene Symbol` `Gene ID` Cytoband `TCGA-19-1790-01B-… `TCGA-41-2572-01A…
## <chr> <dbl> <chr> <dbl> <dbl>
## 1 ENSG0000000812… 0 1p36.33 0 0
## 2 ENSG0000000813… 0 1p36.33 0 0
## 3 ENSG0000006760… 0 1p36.33 0 0
## 4 ENSG0000007836… 0 1p36.33 0 0
## 5 ENSG0000007880… 0 1p36.33 0 0
You can visualize the data using the R/Bioconductor package complexHeatmap.
library(ComplexHeatmap)
library(dplyr)
scores.matrix <- scores[,-c(1:3)] %>% as.matrix
rownames(scores.matrix) <- paste0(scores$`Gene Symbol`,"_",scores$Cytoband)
# gain in more than 100 samples
gain.more.than.hundred.samples <- which(rowSums(scores.matrix == 1) > 100)
# loss in more than 100 samples
loss.more.than.hundred.samples <- which(rowSums(scores.matrix == -1) > 100)
Heatmap(scores.matrix[c(gain.more.than.hundred.samples,loss.more.than.hundred.samples),],
show_column_names = FALSE,
show_row_names = TRUE,
row_names_gp = gpar(fontsize = 8),
col = circlize::colorRamp2(c(-1,0,1), colors = c("red","white","blue")))4.6 DNA methylation data
4.7 ATAC-Seq data
Please, check our ATAC-seq Workshop: http://rpubs.com/tiagochst/atac_seq_workshop
5 Session information
## R version 3.5.3 Patched (2019-03-11 r76979)
## Platform: x86_64-apple-darwin15.6.0 (64-bit)
## Running under: macOS Mojave 10.14.6
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib
##
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
##
## attached base packages:
## [1] grid parallel stats4 stats graphics grDevices utils
## [8] datasets methods base
##
## other attached packages:
## [1] ComplexHeatmap_2.1.0 DT_0.5
## [3] dplyr_0.8.3 maftools_2.0.16
## [5] MultiAssayExperiment_1.8.3 SummarizedExperiment_1.12.0
## [7] DelayedArray_0.8.0 BiocParallel_1.16.6
## [9] matrixStats_0.54.0 Biobase_2.42.0
## [11] GenomicRanges_1.34.0 GenomeInfoDb_1.18.2
## [13] IRanges_2.16.0 S4Vectors_0.20.1
## [15] BiocGenerics_0.28.0 TCGAbiolinks_2.13.4
##
## loaded via a namespace (and not attached):
## [1] backports_1.1.4 circlize_0.4.6
## [3] aroma.light_3.12.0 NMF_0.21.0
## [5] plyr_1.8.4 selectr_0.4-1
## [7] ConsensusClusterPlus_1.46.0 lazyeval_0.2.2
## [9] splines_3.5.3 crosstalk_1.0.0
## [11] ggplot2_3.2.1 gridBase_0.4-7
## [13] sva_3.30.1 digest_0.6.20
## [15] foreach_1.4.7 htmltools_0.3.6
## [17] fansi_0.4.0 magrittr_1.5
## [19] memoise_1.1.0 cluster_2.0.7-1
## [21] doParallel_1.0.15 limma_3.38.3
## [23] Biostrings_2.50.2 readr_1.3.1
## [25] annotate_1.60.1 wordcloud_2.6
## [27] R.utils_2.9.0 prettyunits_1.0.2
## [29] colorspace_1.4-1 blob_1.2.0
## [31] rvest_0.3.4 ggrepel_0.8.1
## [33] xfun_0.8 crayon_1.3.4
## [35] RCurl_1.95-4.12 jsonlite_1.6
## [37] genefilter_1.64.0 zeallot_0.1.0
## [39] survival_2.43-3 zoo_1.8-6
## [41] iterators_1.0.12 glue_1.3.1
## [43] survminer_0.4.5 registry_0.5-1
## [45] gtable_0.3.0 zlibbioc_1.28.0
## [47] XVector_0.22.0 GetoptLong_0.1.7
## [49] shape_1.4.4 scales_1.0.0
## [51] DESeq_1.34.1 rngtools_1.3.1
## [53] DBI_1.0.0 edgeR_3.24.3
## [55] bibtex_0.4.2 ggthemes_4.2.0
## [57] Rcpp_1.0.2 xtable_1.8-4
## [59] progress_1.2.2 clue_0.3-57
## [61] bit_1.1-14 matlab_1.0.2
## [63] km.ci_0.5-2 htmlwidgets_1.3
## [65] httr_1.4.1 RColorBrewer_1.1-2
## [67] pkgconfig_2.0.2 XML_3.98-1.20
## [69] R.methodsS3_1.7.1 utf8_1.1.4
## [71] locfit_1.5-9.1 later_0.8.0
## [73] reshape2_1.4.3 tidyselect_0.2.5
## [75] rlang_0.4.0 AnnotationDbi_1.44.0
## [77] munsell_0.5.0 tools_3.5.3
## [79] cli_1.1.0 downloader_0.4
## [81] generics_0.0.2 RSQLite_2.1.2
## [83] broom_0.5.2 evaluate_0.14
## [85] stringr_1.4.0 yaml_2.2.0
## [87] knitr_1.24 bit64_0.9-7
## [89] survMisc_0.5.5 purrr_0.3.2
## [91] EDASeq_2.16.3 nlme_3.1-137
## [93] mime_0.7 R.oo_1.22.0
## [95] xml2_1.2.2 biomaRt_2.38.0
## [97] compiler_3.5.3 curl_4.0
## [99] png_0.1-7 ggsignif_0.6.0
## [101] tibble_2.1.3 geneplotter_1.60.0
## [103] stringi_1.4.3 GenomicFeatures_1.34.6
## [105] lattice_0.20-38 Matrix_1.2-15
## [107] KMsurv_0.1-5 vctrs_0.2.0
## [109] pillar_1.4.2 GlobalOptions_0.1.0
## [111] data.table_1.12.2 bitops_1.0-6
## [113] httpuv_1.5.1 rtracklayer_1.42.2
## [115] R6_2.4.0 latticeExtra_0.6-28
## [117] hwriter_1.3.2 promises_1.0.1
## [119] ShortRead_1.40.0 gridExtra_2.3
## [121] codetools_0.2-16 assertthat_0.2.1
## [123] pkgmaker_0.27 rjson_0.2.20
## [125] withr_2.1.2 GenomicAlignments_1.18.1
## [127] Rsamtools_1.34.1 GenomeInfoDbData_1.2.0
## [129] mgcv_1.8-27 hms_0.5.0
## [131] prettydoc_0.3.0 tidyr_0.8.3
## [133] rmarkdown_1.14 ggpubr_0.2.2
## [135] shiny_1.3.2
6 Workshop materials
6.1 Workshops HTMLs
- ELMER data Workshop HTML: http://rpubs.com/tiagochst/elmer-data-workshop-2019
- ELMER analysis Workshop HTML: http://rpubs.com/tiagochst/ELMER_workshop
- ATAC-seq Workshop HTML: http://rpubs.com/tiagochst/atac_seq_workshop
6.2 Workshop videos
We have a set of recorded videos, explaining some of the workshops.
- All videos playlist: https://www.youtube.com/playlist?list=PLoDzAKMJh15kNpCSIxpSuZgksZbJNfmMt
- ELMER algorithm: https://youtu.be/PzC31K9vfu0
- ELMER data: https://youtu.be/R00wG--tGo8
- ELMER analysis part1 : https://youtu.be/bcd4uyxrZCw
- ELMER analysis part2: https://youtu.be/vcJ_DSCt4Mo
- ELMER summarizing several analysis: https://youtu.be/moLeik7JjLk
- ATAC-Seq workshop: https://youtu.be/3ftZecz0lU4
References
Colaprico, Antonio, Tiago C Silva, Catharina Olsen, Luciano Garofano, Claudia Cava, Davide Garolini, Thais S Sabedot, et al. 2015. “TCGAbiolinks: An R/Bioconductor Package for Integrative Analysis of Tcga Data.” Nucleic Acids Research 44 (8): e71–e71.